The long-term objective of the proposed work is to achieve a detailed understanding of the mechanisms of pre-mRNA splicing. Splicing plays a essential role in the maturation of most pre-mRNAs in higher eukaryotes, and alternative splicing is involved in regulating the expression of a number of genes. Most pre-mRNAs are highly complex, containing multiple introns that range in size from 65 to 100,000 nucleotides. Thus, elucidating the mechanisms by which each intron is accurately excised, without deleting essential protein coding information is of fundamental biological importance. The main focus of the proposed research is the assembly, structure and function of the spliceosome. Although a great deal of prior work has focused on the identification of the snRNAs involved in splicing, relatively little is known about the protein components of he spliceosome. In the proposed studies, intermediate splicing complexes assembled during the in vitro splicing reaction will be purified and characterized. A two-step method recently established for the large-scale purification of the spliceosome will be employed to isolate each splicing complex, and the RNA and protein components of the complexes will be identified. Complexes assembled on pre-mRNAs containing mutations that block splicing at a specific step of the reaction, as well as intermediate splicing complexes generated during the course of the in vitro splicing reaction ,will be examined. These studies should lead to the identification of splicing factors that interact stably with the pre-mRNA and are involved in different steps of he splicing reaction. In addition ,analysis of intermediate complexes generated very early in the splicing reaction should lead to the identification of factors that play an important role in establishing the pattern of splice-site selection. In subsequent studies, antibodies will be raised against specific proteins that appear to play a central role in splicing. The antibodies will then be used to clone the genes encoding these factors and further defined their function in the splicing reaction. Finally, structural analysis of the spliceosome will be carried out using electron microscopy. These studies analysis of the spliceosome will be carried out using electron microscopy. These studies should provide unique information on the spatial arrangement of pre-mRNA and splicing components within the spliceosome.